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14 pages, 3064 KiB

Open AccessArticle

Inter-Ligand STD NMR: An Efficient 1D NMR Approach to Probe Relative Orientation of Ligands in a Multi-Subsite Protein Binding Pocket

by Serena Monaco, Jonathan Ramírez-Cárdenas, Ana Teresa Carmona, Inmaculada Robina and Jesus Angulo

Pharmaceuticals 2022, 15(8), 1030; https://doi.org/10.3390/ph15081030 - 21 Aug 2022

Cited by 6 | Viewed by 2786

Abstract

In recent years, Saturation Transfer Difference NMR (STD NMR) has been proven to be a powerful and versatile ligand-based NMR technique to elucidate crucial aspects in the investigation of protein-ligand complexes. Novel STD NMR approaches relying on “multi-frequency” irradiation have enabled us to [...] Read more.

In recent years, Saturation Transfer Difference NMR (STD NMR) has been proven to be a powerful and versatile ligand-based NMR technique to elucidate crucial aspects in the investigation of protein-ligand complexes. Novel STD NMR approaches relying on “multi-frequency” irradiation have enabled us to even elucidate specific ligand-amino acid interactions and explore the binding of fragments in previously unknown binding subsites. Exploring multi-subsite protein binding pockets is especially important in Fragment Based Drug Discovery (FBDD) to design leads of increased specificity and efficacy. We hereby propose a novel multi-frequency STD NMR approach based on direct irradiation of one of the ligands in a multi-ligand binding process, to probe the vicinity and explore the relative orientation of fragments in adjacent binding sub-sites, which we called Inter-Ligand STD NMR (IL-STD NMR). We proved its applicability on (i) a standard protein-ligand system commonly used for ligand-observed NMR benchmarking: Naproxen as bound to Bovine Serum Albumin, and (ii) the biologically relevant system of Cholera Toxin Subunit B and two inhibitors adjacently bound within the GM1 binding site. Relative to Inter-Ligand NOE (ILOE), the current state-of-the-art methodology to probe relative orientations of adjacent ligands, IL-STD NMR requires about one tenth of the experimental time and protein consumption, making it a competitive methodology with the potential to be applied in the pharmaceutical industries. Full article

(This article belongs to the Special Issue High Field NMR and Ultra-High Field NMR in Medicinal Chemistry)

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10 pages, 3435 KiB

Open AccessArticle

Pleiotrophin Interaction with Synthetic Glycosaminoglycan Mimetics

by Jonathan R. Miles, Xu Wang, Jose L. de Paz and Pedro M. Nieto

Pharmaceuticals 2022, 15(5), 496; https://doi.org/10.3390/ph15050496 - 19 Apr 2022

Viewed by 1577

Abstract

Chondroitin sulfate (CS) E is the natural ligand for pleiotrophin (PTN) in the central nervous system (CNS) of the embryo. Some structures of PTN in solution have been solved, but no precise location of the binding site has been reported yet. Using ¹⁵ [...] Read more.

Chondroitin sulfate (CS) E is the natural ligand for pleiotrophin (PTN) in the central nervous system (CNS) of the embryo. Some structures of PTN in solution have been solved, but no precise location of the binding site has been reported yet. Using ¹⁵N-labelled PTN and HSQC NMR experiments, we studied the interactions with a synthetic CS-E tetrasaccharide corresponding to the minimum binding sequence. The results agree with the data for larger GAG (glycosaminoglycans) sequences and confirm our hypothesis that a synthetic tetrasaccharide is long enough to fully interact with PTN. We hypothesize that the central region of PTN is an intrinsically disordered region (IDR) and could modify its properties upon binding. The second tetrasaccharide has two benzyl groups and shows similar effects on PTN. Finally, the last measured compound aggregated but beforehand, showed a behavior compatible with a slow exchange in the NMR time scale. We propose the same binding site and mode for the tetrasaccharides with and without benzyl groups. Full article

(This article belongs to the Special Issue High Field NMR and Ultra-High Field NMR in Medicinal Chemistry)

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17 pages, 5407 KiB

Open AccessArticle

Oligosaccharide Presentation Modulates the Molecular Recognition of Glycolipids by Galectins on Membrane Surfaces

by Marta G. Lete, Antonio Franconetti, Sandra Delgado, Jesús Jiménez-Barbero and Ana Ardá

Pharmaceuticals 2022, 15(2), 145; https://doi.org/10.3390/ph15020145 - 26 Jan 2022

Cited by 3 | Viewed by 2871

Abstract

Galectins are a family of glycan binding proteins that stand out for the wide range of biological phenomena in which they are involved. Most galectin functions are associated with their glycan binding capacities, which are generally well characterized at the oligosaccharide level, but [...] Read more.

Galectins are a family of glycan binding proteins that stand out for the wide range of biological phenomena in which they are involved. Most galectin functions are associated with their glycan binding capacities, which are generally well characterized at the oligosaccharide level, but not at the glycoprotein or glycolipid level. Glycolipids form the part of cell membranes where they can act as galectin cellular receptors. In this scenario, glycan presentation as well as the membrane chemical and structural features are expected to have a strong impact in these molecular association processes. Herein, liposomes were used as membrane mimicking scaffolds for the presentation of glycosphingolipids (GSLs) and to investigate their interaction with Galectin-3 and the N-domain of Galectin-8 (Gal8N). The binding towards GM3 and GM1 and their non-silaylated GSLs was compared to the binding to the free glycans, devoid of lipid. The analysis was carried out using a combination of NMR methods, membrane perturbation studies, and molecular modeling. Our results showed a different tendency of the two galectins in their binding capacities towards the glycans, depending on whether they were free oligosaccharides or as part of GSL inserted into a lipid bilayer, highlighting the significance of GSL glycan presentation on membranes in lectin binding. Full article

(This article belongs to the Special Issue High Field NMR and Ultra-High Field NMR in Medicinal Chemistry)

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18 pages, 4567 KiB

Open AccessArticle

Zinc Binding to NAP-Type Neuroprotective Peptides: Nuclear Magnetic Resonance Studies and Molecular Modeling

by Ancuta-Veronica Lupaescu, Cosmin Stefan Mocanu, Gabi Drochioiu and Catalina-Ionica Ciobanu

Pharmaceuticals 2021, 14(10), 1011; https://doi.org/10.3390/ph14101011 - 01 Oct 2021

Cited by 5 | Viewed by 2108

Abstract

Aggregation of amyloid-β peptides (Aβ) is a hallmark of Alzheimer’s disease (AD), which is affecting an increasing number of people. Hence, there is an urgent need to develop new pharmaceutical treatments which could be used to prevent the AD symptomatology. Activity-dependent neuroprotective protein [...] Read more.

Aggregation of amyloid-β peptides (Aβ) is a hallmark of Alzheimer’s disease (AD), which is affecting an increasing number of people. Hence, there is an urgent need to develop new pharmaceutical treatments which could be used to prevent the AD symptomatology. Activity-dependent neuroprotective protein (ADNP) was found to be deficient in AD, whereas NAP, an 8-amino-acid peptide (¹NAPVSIPQ⁸) derived from ADNP, was shown to enhance cognitive function. The higher tendency of zinc ion to induce Aβ aggregation and formation of amorphous aggregates is also well-known in the scientific literature. Although zinc binding to Aβ peptides was extensively investigated, there is a shortage of knowledge regarding the relationship between NAP peptide and zinc ions. Therefore, here, we investigated the binding of zinc ions to the native NAP peptide and its analog obtained by replacing the serine residue in the NAP sequence with tyrosine (¹NAPVYIPQ⁸) at various molar ratios and pH values by mass spectrometry (MS) and nuclear magnetic resonancespectroscopy (NMR). Matrix-assisted laser desorption/ionization time-of-flight (MALDI ToF) mass spectrometry confirmed the binding of zinc ions to NAP peptides, while the chemical shift of Asp¹, observed in ¹H-NMR spectra, provided direct evidence for the coordinating role of zinc in the N-terminal region. In addition, molecular modeling has also contributed largely to our understanding of Zn binding to NAP peptides. Full article

(This article belongs to the Special Issue High Field NMR and Ultra-High Field NMR in Medicinal Chemistry)

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25 pages, 3571 KiB

Open AccessArticle

Semaphorin 3A—Glycosaminoglycans Interaction as Therapeutic Target for Axonal Regeneration

by Yolanda Pérez, Roman Bonet, Miriam Corredor, Cecilia Domingo, Alejandra Moure, Àngel Messeguer, Jordi Bujons and Ignacio Alfonso

Pharmaceuticals 2021, 14(9), 906; https://doi.org/10.3390/ph14090906 - 07 Sep 2021

Cited by 4 | Viewed by 3364

Abstract

Semaphorin 3A (Sema3A) is a cell-secreted protein that participates in the axonal guidance pathways. Sema3A acts as a canonical repulsive axon guidance molecule, inhibiting CNS regenerative axonal growth and propagation. Therefore, interfering with Sema3A signaling is proposed as a therapeutic target for achieving [...] Read more.

Semaphorin 3A (Sema3A) is a cell-secreted protein that participates in the axonal guidance pathways. Sema3A acts as a canonical repulsive axon guidance molecule, inhibiting CNS regenerative axonal growth and propagation. Therefore, interfering with Sema3A signaling is proposed as a therapeutic target for achieving functional recovery after CNS injuries. It has been shown that Sema3A adheres to the proteoglycan component of the extracellular matrix (ECM) and selectively binds to heparin and chondroitin sulfate-E (CS-E) glycosaminoglycans (GAGs). We hypothesize that the biologically relevant interaction between Sema3A and GAGs takes place at Sema3A C-terminal polybasic region (SCT). The aims of this study were to characterize the interaction of the whole Sema3A C-terminal polybasic region (Sema3A 725–771) with GAGs and to investigate the disruption of this interaction by small molecules. Recombinant Sema3A basic domain was produced and we used a combination of biophysical techniques (NMR, SPR, and heparin affinity chromatography) to gain insight into the interaction of the Sema3A C-terminal domain with GAGs. The results demonstrate that SCT is an intrinsically disordered region, which confirms that SCT binds to GAGs and helps to identify the specific residues involved in the interaction. NMR studies, supported by molecular dynamics simulations, show that a new peptoid molecule (CSIC02) may disrupt the interaction between SCT and heparin. Our structural study paves the way toward the design of new molecules targeting these protein–GAG interactions with potential therapeutic applications. Full article

(This article belongs to the Special Issue High Field NMR and Ultra-High Field NMR in Medicinal Chemistry)

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11 pages, 2126 KiB

Open AccessArticle

Exploring the Early Stages of the Amyloid Aβ(1–42) Peptide Aggregation Process: An NMR Study

by Angelo Santoro, Manuela Grimaldi, Michela Buonocore, Ilaria Stillitano and Anna Maria D’Ursi

Pharmaceuticals 2021, 14(8), 732; https://doi.org/10.3390/ph14080732 - 27 Jul 2021

Cited by 12 | Viewed by 2930

Abstract

Alzheimer’s disease (AD) is a neurodegenerative pathology characterized by the presence of neurofibrillary tangles and amyloid plaques, the latter mainly composed of Aβ(1–40) and Aβ(1–42) peptides. The control of the Aβ aggregation process as a therapeutic strategy for AD has prompted the interest [...] Read more.

Alzheimer’s disease (AD) is a neurodegenerative pathology characterized by the presence of neurofibrillary tangles and amyloid plaques, the latter mainly composed of Aβ(1–40) and Aβ(1–42) peptides. The control of the Aβ aggregation process as a therapeutic strategy for AD has prompted the interest to investigate the conformation of the Aβ peptides, taking advantage of computational and experimental techniques. Mixtures composed of systematically different proportions of HFIP and water have been used to monitor, by NMR, the conformational transition of the Aβ(1–42) from soluble α-helical structure to β-sheet aggregates. In the previous studies, 50/50 HFIP/water proportion emerged as the solution condition where the first evident Aβ(1–42) conformational changes occur. In the hypothesis that this solvent reproduces the best condition to catch transitional helical-β-sheet Aβ(1–42) conformations, in this study, we report an extensive NMR conformational analysis of Aβ(1–42) in 50/50 HFIP/water v/v. Aβ(1–42) structure was solved by us, giving evidence that the evolution of Aβ(1–42) peptide from helical to the β-sheet may follow unexpected routes. Molecular dynamics simulations confirm that the structural model we calculated represents a starting condition for amyloid fibrils formation. Full article

(This article belongs to the Special Issue High Field NMR and Ultra-High Field NMR in Medicinal Chemistry)

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20 pages, 2689 KiB

Open AccessSystematic Review

Pharmacometabolomics by NMR in Oncology: A Systematic Review

by Nuria Gómez-Cebrián, Pedro Vázquez Ferreiro, Francisco Javier Carrera Hueso, José Luis Poveda Andrés, Leonor Puchades-Carrasco and Antonio Pineda-Lucena

Pharmaceuticals 2021, 14(10), 1015; https://doi.org/10.3390/ph14101015 - 02 Oct 2021

Cited by 4 | Viewed by 2853

Abstract

Pharmacometabolomics (PMx) studies aim to predict individual differences in treatment response and in the development of adverse effects associated with specific drug treatments. Overall, these studies inform us about how individuals will respond to a drug treatment based on their metabolic profiles obtained [...] Read more.

Pharmacometabolomics (PMx) studies aim to predict individual differences in treatment response and in the development of adverse effects associated with specific drug treatments. Overall, these studies inform us about how individuals will respond to a drug treatment based on their metabolic profiles obtained before, during, or after the therapeutic intervention. In the era of precision medicine, metabolic profiles hold great potential to guide patient selection and stratification in clinical trials, with a focus on improving drug efficacy and safety. Metabolomics is closely related to the phenotype as alterations in metabolism reflect changes in the preceding cascade of genomics, transcriptomics, and proteomics changes, thus providing a significant advance over other omics approaches. Nuclear Magnetic Resonance (NMR) is one of the most widely used analytical platforms in metabolomics studies. In fact, since the introduction of PMx studies in 2006, the number of NMR-based PMx studies has been continuously growing and has provided novel insights into the specific metabolic changes associated with different mechanisms of action and/or toxic effects. This review presents an up-to-date summary of NMR-based PMx studies performed over the last 10 years. Our main objective is to discuss the experimental approaches used for the characterization of the metabolic changes associated with specific therapeutic interventions, the most relevant results obtained so far, and some of the remaining challenges in this area. Full article

(This article belongs to the Special Issue High Field NMR and Ultra-High Field NMR in Medicinal Chemistry)

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15 pages, 1673 KiB

Open AccessConcept Paper

Real-Time Non-Invasive and Direct Determination of Lactate Dehydrogenase Activity in Cerebral Organoids—A New Method to Characterize the Metabolism of Brain Organoids?

by Gal Sapir, Daniel J. Steinberg, Rami I. Aqeilan and Rachel Katz-Brull

Pharmaceuticals 2021, 14(9), 878; https://doi.org/10.3390/ph14090878 - 30 Aug 2021

Cited by 2 | Viewed by 2882

Abstract

Organoids are a powerful tool in the quest to understand human diseases. As the developing brain is extremely inaccessible in mammals, cerebral organoids (COs) provide a unique way to investigate neural development and related disorders. The aim of this study was to utilize [...] Read more.

Organoids are a powerful tool in the quest to understand human diseases. As the developing brain is extremely inaccessible in mammals, cerebral organoids (COs) provide a unique way to investigate neural development and related disorders. The aim of this study was to utilize hyperpolarized ¹³C NMR to investigate the metabolism of COs in real-time, in a non-destructive manner. The enzymatic activity of lactate dehydrogenase (LDH) was determined by quantifying the rate of [1-¹³C]lactate production from hyperpolarized [1-¹³C]pyruvate. Organoid development was assessed by immunofluorescence imaging. Organoid viability was confirmed using ³¹P NMR spectroscopy. A total of 15 organoids collated into 3 groups with a group total weight of 20–77 mg were used in this study. Two groups were at the age of 10 weeks and one was at the age of 33 weeks. The feasibility of this approach was demonstrated in both age groups, and the LDH activity rate was found to be 1.32 ± 0.75 nmol/s (n = 3 organoid batches). These results suggest that hyperpolarized NMR can be used to characterize the metabolism of brain organoids with a total tissue wet weight of as low as 20 mg (<3 mm³) and a diameter ranging from 3 to 6 mm. Full article

(This article belongs to the Special Issue High Field NMR and Ultra-High Field NMR in Medicinal Chemistry)

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